Project Summary: The goal of this proposal is to develop a significantly safer variant of the anti-cancer biologic drug L- asparaginase (ASNase). ASNases are enzyme drugs that systematically deplete L-asparagine from the blood. First generation ASNases are approved by the FDA for the treatment of acute lymphoblastic leukemia (ALL), a cancer of white blood cells. This generation, including enzymes from E. coli (Elspar) and Erwinia chrysanthemi (Erwinaze), is severely hampered by toxicity which not only reduces the quality of life for patients, but whose effects can be so severe as to be fatal. Side effects stem from two causes ? one coming from the L-glutaminase coactivity of these enzyme drugs and the second from the immunogenicity due to their bacterial origin. To address the immunogenic side effects, second generation ASNases are PEGylated. They include the PEGylated E. coli enzyme (Oncaspar), which is currently the standard of care in the USA, and the PEGylated Erwinia enzyme (Asparec), which is currently in clinical trials. Whereas PEGylation reduces but does not eliminate immunogenicity problems, the side effects caused by their high L- glutaminase coactivity remains a major clinical challenge. Despite ASNases being key drugs in pediatric ALL treatment, these side effects are so pronounced in adults that their use is largely avoided. This factors into the much lower cure rate (~40%) of the adult ALL population. Additionally, ASNase-associated side effects prevent the use of this unique cancer drug in other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. pancreatic cancer), despite strong evidence that ASNases would be effective in treating those cancers. Hence, there is a clear unmet need for an ASNase with reduced immunogenicity and that lacks L-glutaminase coactivity. Recently, we characterized a guinea pig ASNase (GpA) that possess the required low KM property for clinical efficacy and that has exhibited in vivo tumor cell-killing. Notably, we also discovered that GpA is devoid of the toxicity-causing L-glutaminase coactivity. With ~70% sequence identity to human ASNase, GpA should be less immunogenic compared to the bacterial enzymes that share only ~25% sequence identity with the human enzyme. We recently used a genetic screen to identify highly humanized (~86% identity) GpA variants and identified a stable and active C-terminal truncation of GpA comprising the catalytic domain. We call this variant 63N. We also employed structural information to identify surface residues on 63N that can be mutated into cysteines and are appropriately spaced to permit site-specific PEGylation that results in masking the drug from the immune system as well as proteases. This technology is superior to that used in second generation enzymes where lysine PEGylation results in a non-homogenous product, variations between batches, and reduced enzymatic (i.e. drug) activity. An additional advantage of PEGylation is increased in vivo stability that will allow lower dosing at more extended intervals. The goal of this Phase I STTR application is to develop a proof of concept for such a third generation ASNase drug.